1. Field of the Invention
The present invention relates to a composition containing 9-cis .beta.-carotene in high-purity and a method of obtaining the same.
2. Related Arts
.beta.-Carotene is one of carotenoids contained in various colored vegetables and so on and has been well known as a substance which changes to vitamin A in vivo to show high biological activity. Further, .beta.-carotene has been highly expected as one of ingredients for medicines, since it shows an anti-tumor activity and has an ability of eliminating active oxygen.
Natural .beta.-carotene is mainly of all-trans type but various geometrical isomers thereof present, and in recent years, activities of the isomers have been greatly interested, since such isomers have been detected in human tissue, in addition to all-trans .beta.-carotene.
Especially, 9-cis .beta.-carotene becomes a center of such an attention, among the isomers. Since 9-cis .beta.-carotene has been produced and accumulated in relatively large amount in green algae belonging to microalgae, Dunaliella, so that extractions of the substance from such a source have been tried [U.S. Pat. No. 5,310,554; U.S. Pat. No. 5,612,485; and Yuan et al., "Yaowu Shengwu Jishu", Vol. 3, pages 34-39 (1996) and so on].
However, almost all of the conventional methods of obtaining the composition containing 9-cis .beta.-carotene utilize column chromatography and thus are not suitable for industrial scale production of the composition. The inventors have also tried to extract 9-cis .beta.-carotene from Dunaliella bardawil by using hexane and obtained a solid composition containing 9-cis .beta.-carotene, but content of 9-cis .beta.-carotene therein remarkably varies in each experiment, so that they applied many complicated procedures including column chromatography to obtain fine needle-shaped 9-cis .beta.-carotene as follows: removing the solid materials in hexane to obtain a filtrate, subjecting the filtrate to a silica gel chromatography, concentrating a carotene-rich fraction to obtain foamy solids, sonicating the solids in ethanol to obtain orange particles, dissolving the particles into chloroform, subjecting the resulting solution to ODS open column chromatography, concentrating a carotene-rich fraction to obtain an orange powder, subjecting the powder to preparative high-performance liquid chromatography (HPLC), and then concentrating the HPLC fraction which contains 9-cis .beta.-carotene to cause precipitation thereof as the crystals [Suzuki et al., "Biochem. Mol. Biol. Int.", Vol. 39, pages 1077-1084 (1996)].
Methods of obtaining 9-cis .beta.-carotene utilizing no column chromatography have also been proposed, but all of reports on such proposals have so referred to that a composition obtained is oily substance, in spite of that 9-cis .beta.-carotene, inherently, is a solid substance having melting point of 122-123.degree. C. [Polgar et al., "J. Amer. Chem. Soc.", Vol. 64, pages 1856-1861 (1942)]. This means that each of the compositions contains impurities other than 9-cis .beta.-carotene, in some extent. In such a report, it has oftenly been described that "high-purity 9-cis .beta.-carotene" was obtained by referring to its ratio to total carotene, but this report has some doubt in that impurities other than carotenes have not been taken into consideration and this becomes a great problem in case of that the composition shall be actually used as an ingredient for medicines.